cancers Review Animal Models: A Useful Tool to Unveil Metabolic Changes in Hepatocellular Carcinoma Marina Serra, Amedeo Columbano , Andrea Perra * and Marta Anna Kowalik * Unit of Oncology and Molecular Pathology, Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato, Italy; [email protected] (M.S.); [email protected] (A.C.) * Correspondence: [email protected] (A.P.); [email protected] (M.A.K.) Received: 27 September 2020; Accepted: 8 November 2020; Published: 10 November 2020 Simple Summary: Hepatocellular carcinoma (HCC) represents an important health problem. At the moment, systemic therapies offered only modest clinical benefits. Thus, HCC represents a cancer extremely difficult to treat, and therapeutic breakthroughs are urgently needed. Metabolic reprogramming of neoplastic cells has been recognized as one of the core hallmarks of cancer. Experimental animal models represent an important tool that allows to investigate metabolic changes underlying HCC development and progression. In the present review, we characterize available rodent models of hepatocarcinogenesis. Moreover, we discuss the possibility that pharmacological targeting of Warburg metabolism may represent an additional tool to improve already available therapeutic approaches for HCC. Abstract: Hepatocellular carcinoma (HCC) is one the most frequent and lethal human cancers. At present, no effective treatment for advanced HCC exist; therefore, the overall prognosis for HCC patients remains dismal. In recent years, a better knowledge of the signaling pathways involved in the regulation of HCC development and progression, has led to the identification of novel potential targets for therapeutic strategies. However, the obtained benefits from current therapeutic options are disappointing. Altered cancer metabolism has become a topic of renewed interest in the last decades, and it has been included among the core hallmarks of cancer. In the light of growing evidence for metabolic reprogramming in cancer, a wide number of experimental animal models have been exploited to study metabolic changes characterizing HCC development and progression and to further expand our knowledge of this tumor. In the present review, we discuss several rodent models of hepatocarcinogenesis, that contributed to elucidate the metabolic profile of HCC and the implications of these changes in modulating the aggressiveness of neoplastic cells. We also highlight the apparently contrasting results stemming from different animal models. Finally, we analyze whether these observations could be exploited to improve current therapeutic strategies for HCC. Keywords: HCC; glycolysis; OXPHOS; PPP; miRNA 1. Introduction 1.1. Hepatocellular Carcinoma (HCC) Primary liver cancer, which includes predominantly hepatocellular carcinoma (HCC) (75–85% of cases), ranks third in terms of mortality worldwide, and represents an important challenge for global health [1]. Regions with high or low-rate incidence of HCC are characterized by a prevalence of different risk factors. Importantly, most HCCs develop in patients with underlying liver cirrhosis and are associated with different etiologies, including hepatitis B virus (HBV) or hepatitis C virus (HCV) Cancers 2020, 12, 3318; doi:10.3390/cancers12113318 www.mdpi.com/journal/cancers Cancers 2020, 12, 3318 2 of 27 chronic infection, exposure to carcinogenic compounds like aflatoxin B1, alcoholic and non-alcoholic fatty liver disease (AFLD and NAFLD), smoking and type 2 diabetes [2]. Presently, there are only a few drugs approved by Food and Drug Administration (FDA). The first is Sorafenib, an oral multikinase inhibitor of the vascular endothelial growth factor receptor (VEGRF), the platelet-derived growth factor receptor (PDGFR) and Raf for the treatment of unresectable HCC. However, an increased survival of only 3 months was shown in the Sorafenib Hepatocellular Carcinoma Assessment Randomized Protocol (SHARP) among patients with advanced disease [3]. A similar slight improvement was obtained when sorafenib treatment was followed by regorafenib, another tyrosine kinase (TK) inhibitor [4]. New therapeutic strategies, such as Atezolizumab plus Bevacizumab for advanced and metastatic HCC together with Lenvatinib as the frontline line treatment option, followed by Cabozantinib and Ramucirumab as the second line targeted agents have been recently introduced [5,6]. In addition, immunotherapy has been proposed for HCC treatment, based on the finding that the programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) axis is an adaptive immune resistance mechanism used by cancer cells to overcome immune anti-tumor activity. Several studies have demonstrated that overexpression of PD-L1 is associated with more aggressive phenotype and worse prognosis of HCC [7–9]. Accordingly, nivolumab, an inhibitor of PD-1 exhibited positive results in patients with advanced HCC with or without chronic viral hepatitis [10], although the overall therapeutic effect remains still unsatisfactory. However, the results are not satisfactory, and no effective systemic therapy exists for patients with advanced HCC. A more complete understanding of the molecular mechanisms involved in HCC development and progression thus is required to improve therapeutic strategies against this lethal cancer. Over the last years, many fundamental studies have explored genomic alterations and identified the most frequently mutated genes in HCC. Mutations of the TERT promoter able to activate telomerase expression, have been described as the earliest alterations in HCC development, as they were found in a significant percentage of dysplastic nodules [11], while molecular alterations in cell cycle control (TP53, RB1, CCND1, CDKN2A), Wnt/β-catenin signaling (CTNNB1, AXIN1), oxidative stress response (NFE2L2, KEAP1), epigenetic regulation (ARID1A, ARID2) and the protein kinase B/mammalian target of rapamycin (AKT/mTOR) and mitogen-activated protein (MAP) kinase pathway were shown to take place at late stages of tumorigenesis [12–16]. More recently, epigenetic events, such as methylation status, have been argued to play a critical role in HCC progression [17]. 1.2. Metabolic Changes Observed in Human HCC Oncogenic mutations cause alterations to numerous signaling pathways that affect tumor cell metabolism and lead to enhanced survival and growth [18,19]. Parallel, though limited, studies have investigated the presence and possible significance of metabolic alterations in HCC, engendering new insight into this cancer type. Worth of reminding is that a marked interest in tumor metabolism has been observed since almost a century, when aerobic glycolysis or ‘Warburg phenomenon/effect’ [20,21] was described. This metabolic shift, reporting that most cancer cells enhance glucose utilization independently of oxygen availability, has now achieved the status of a core hallmark of cancer [22]. The switch from oxidative phosphorylation (OXPHOS)—the most efficient pathway for producing adenosine triphosphate (ATP) to glycolysis observed in cancer cells is likely required to withstand the increased anabolic demands of tumor cells [23,24]. Such needs of cancer cells are satisfied mainly by conveying metabolites into the pentose phosphate pathway (PPP). PPP provides both ribose-5-phosphate, a structural component of nucleotides, to sustain cell proliferation and reduced nicotinamide adenine dinucleotide phosphate (NADPH), utilized for fatty acid and cholesterol biosynthesis, as well as required for the generation of reduced glutathione (GSH), a major scavenger of reactive oxygen species. These processes are regulated primarily by the action of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP [25–27]. The metabolic switch from OXPHOS to glycolysis allows neoplastic cells to enhance their biosynthetic capabilities by expressing a tumor-specific form of pyruvate kinase (PK). This catalyzes the rate-limiting, Cancers 2020, 12, 3318 3 of 27 ATP-generating, irreversible reaction of glycolysis, the conversion of phosphoenolpyruvate (PEP) to pyruvate [28–30]. The M2 isoform of pyruvate kinase (PKM2) of cancer cells is under control of numerous allosteric modulators that can activate or inhibit its activity [28,29] and a switch between a highly active tetrameric form of PKM2 and an almost inactive dimeric form is considered as one of the main regulators of Warburg metabolism [31]. Inhibition of PKM2 can also increase levels of glucose-6-phosphate in order to increase oxidative PPP flux [32]. With regard to HCC, different aspects of metabolic reprogramming have been investigated over the last years [33]. Firstly,metabolomics, which summarizes the metabolic status of the organism and reflects the dynamic interactions between genes, proteins and the environment [34,35], showed an alteration of energy metabolism between HCC and adjacent non-involved tissues [36]. Successively, combined transcriptomic and metabolomic studies of energy metabolism in HCC demonstrated an increase in glycolysis over mitochondrial OXPHOS [37], as also recently reported [16]. In particular, OXPHOS was shown to be among the most significantly downregulated pathways in a homogeneous HCC subgroup driven by the activation of cyclin A2 (CCNA2) or cyclin E1 (CCNE1) gene [38]. A recent thorough study of the expression of metabolic genes in human HCCs [39] reported more than 600 consistently altered metabolic genes, mostly involved in glycolysis, PPP, nucleotide biosynthesis, tricarboxylic acid (TCA) cycle, OXPHOS,